1. Field of the Invention
This invention relates to novel, heat-resistant sarcosine oxidase N and a process for producing the same.
2. Description of the Prior Art
Sarcosine oxidase is an enzyme which catalyzes the following reaction. EQU Sarcosine+H.sub.2 O+O.sub.2 .fwdarw.glycine+formaldehyde+H.sub.2 O.sub.2
Sarcosine oxidase is known and described in, for example, U.S. Pat. No. 4,216,292 and J. Biochem. 89, 599 (1981), JAPAN.
Meanwhile, in the field of clinical medicine, quantitative determination of creatinine or creatine present in serum and urine is currently conducted for examination of renal function disorder, muscular disorder, etc. For this determination, there is known a so-called Jaffe method wherein creatinine is reacted with alkaline picric acid and the resulting orange color is measured. This color-developing reaction is affected by various substances present in serum and urine and accordingly is a non-specific reaction. Consequently, the value obtained by the Jaffe method is low in reliability. Moreover, since a step for protein removal is necessary in many cases, the method is troublesome in procedure.
A method for enzymatically determining creatinine or creatine has recently been reported [Clinical Science Symposium No. 19, 196 (1979)]. This method uses sarcosine oxidase as shown below. In the following formulas, the enzymes used are given in parentheses. EQU Creatinine+H.sub.2 O.revreaction.creatine
(Creatinine amidohydrolase) EQU Creatine+H.sub.2 O.fwdarw.sarcosine+urea PA2 (Creatine amidinohydrolase) EQU Sarcosine+H.sub.2 O+O.sub.2 .fwdarw.glycine+formaldehyde+H.sub.2 O.sub.2 PA2 (Sarcosine oxidase)
However, since the amount of creatinine or creatine present in serum and urine is very small, there has been desired the development of a high sensitivity method for enzymatic determination of creatinine or creatine.
A coloring agent has recently been developed which develops a color at a high sensitivity when hydrogen peroxide (H.sub.2 O.sub.2) is treated, under neutrality or slight acidity, with a peroxidase obtained from horseradish. This color is blue or green and is different from yellow or red originated from serum components; therefore, no blank measurement for serum is necessary, which is advantageous. However, the above color is unstable and disappears at pH 7.5 or above. Consequently, it is necessary to conduct the enzymatic reaction/color-developing reaction under neutrality or slight acidity to expect a stable color-developing reaction. Moreover, since the optimum pH of peroxidase is at or around 6.5 (slight acidic), the coupled enzymes are also required to have a satisfactory enzymatic action under slight acidity.
The enzymes used for determination of creatinine or creatine are required to be highly pure and free from other accompanying enzymes. Therefore, there is desired the development of enzymes of high relative activity and high purity which can be obtained from a mass purification step wherein other accompanying enzymes (e.g. catalase and uricase) are completely inactivated by a heat treatment.
Since the amount of creatinine or creatine contained in serum and urine is small, the amount of sarcosine formed from the enzymatic decomposition of creatinine or creatine is inevitably small. Therefore, there is strongly desired in the industry the development of an enzyme which can act on even such a small amount of sarcosine and has a small Km value (Michaelis constant).
These tasks have been achieved by the present invention.
In view of the above situation, the present inventors conducted an extensive study in order to provide a novel sarcosine oxidase suitable for high sensitivity enzymatic determination of creatinine or creatine which has a satisfactory enzymatic action even under slight acidity, is thermally stable and has a low Km value for sarcosine. As a result, it was found that by culturing a strain belonging to genus Bacillus, newly isolated from the soil, there can be obtained a novel sarcosine oxidase having a satisfactory enzymatic activity even under slight acidity, being heat-resistant and having a low Km value for sarcosine. This finding has led to the completion of the present invention.